Anna Riccioli

Testicular FasL is expressed by sperm cells

The testis is the main source of Fas ligand (FasL) mRNA in rodents; it is generally believed that this molecule, expressed on bordering somatic Sertoli cells, bestows an immune-privileged status in the testis by eliminating infiltrating inflammatory Fas-bearing leukocytes. Our results demonstrate that the attribution of testicular expression of FasL to Sertoli cells is erroneous and that FasL transcription instead occurs in meiotic and postmeiotic germ cells, whereas the protein is only displayed on mature spermatozoa. These findings point to a significant role of the Fas system in the biology of mammalian reproduction.

Toll-like receptor 3 triggers apoptosis of human prostate cancer cells through a PKC-α-dependent mechanism

Toll-like receptors (TLRs) are known to play a key role in the innate immune system particularly in inflammatory response against invading pathogens. Recent reports strongly indicate that they play important roles in cancer cells. Prostate cancer represents one of the most common cancer for which no cure is available once metastatic and androgen refractory. Since TLR3 has been recently suggested as a possible therapeutic target in some cancer cell lines, we studied TLR3 expression and functionality in two human prostate cancer cell lines, LNCaP and PC3. We report that both cell lines express TLR3 and that the TLR3 agonist poly (I:C) activates mitogen-activated protein kinases and induces inhibition of proliferation as well as caspase-dependent apoptosis. By using pharmacological and genetic approaches, we demonstrate the involvement of TLR3 in poly (I:C)-induced effects. We also show that a novel interferon-independent pathway involving protein kinase C (PKC)-alpha activation, upstream of p38 and c-jun N-terminal kinase, is responsible for poly (I:C) pro-apoptotic effects on LNCaP cells. To our knowledge, this is the first report describing a role of PKC-alpha in poly (I:C)-mediated apoptosis. The comprehension of the mechanisms underlying TLR3-mediated apoptosis can contribute tools to develop new agonists useful for the treatment of prostate cancer.

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-α and IFN-γ markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-α-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-α and IFN-γ on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.

Immunosuppressive molecules produced by Sertoli cells cultured in vitro : biological effects on lymphocytes

In the present study we have analyzed the proteins secreted in vitro by murine Sertoli cells to identify immunosuppressive factors. Our data show that Sertoli cells secrete molecules capable to inhibit proliferation of lymphocytes activated in vitro. Cytophluorimetric analysis indicates that treated cells are arrested in the G1 phase of cell cycle. The inhibitory activity is specific for both B or T lymphocytes but not for other non-lymphoid cells and is associated to proteins, heat and freeze stable, with Mr of more than 30 kDa. Lymphocytes treated with Sertoli immunosuppressive proteins drastically reduce the secretion of interleukin-2.

Sertoli Cells Initiate Testicular Innate Immune Responses through TLR Activation

TLRs play a crucial role in early host defense against invading pathogens. In the seminiferous epithelium, Sertoli cells are the somatic nurse cells that mechanically segregate germ cell autoantigens by means of the blood-tubular barrier and create a microenvironment that protects germ cells from both interstitial and ascending invading pathogens. The objective of this study was to examine TLR expression and their functional responses to specific agonists in mouse Sertoli cells. We measured the expression of TLR2, TLR4, TLR5, and TLR6 mRNAs and confirmed by FACS analysis the presence of proteins TLR2 and TLR5 on which we focused our study. Stimulation of Sertoli cells with macrophage-activating lipopeptide-2, agonist of TLR2/TLR6, and with flagellin, agonist of TLR5, induces augmented secretion of the chemokine MCP-1. To assess the functional significance of MCP-1 production following TLR stimulation, conditioned medium from either macrophage-activating lipopeptide-2 or flagellin-treated Sertoli cells was tested for in vitro chemotaxis assay, and a significant increase of macrophage migration was observed in comparison with unstimulated conditioned medium. Moreover, we studied the role of NF-κB and of MAPKs in regulating TLR-mediated MCP-1 secretion by using inhibitors specific for each transduction pathway and we demonstrated a pivotal role of the IκB/NF-κB and JNK systems. In addition, TLR2/TLR6 and TLR5 stimulation induces increased ICAM-1 expression in Sertoli cells. Collectively, this study demonstrates the novel ability of Sertoli cells to potentially respond to a wide variety of bacteria through TLR stimulation.

Toll-Like Receptor 3 Activation Induces Antiviral Immune Responses in Mouse Sertoli Cells

Abstract Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and elicit antimicrobial immune responses. In the testis, viruses can induce pathological conditions, such as orchitis, and may participate in the etiology of testicular cancer; however, the molecular mechanisms involved remain under investigation. It has been suggested that because they constitutively express interferon (IFN)-inducible antiviral proteins, Sertoli cells participate in the testicular antiviral defense system. Previously, we demonstrated a key function of mouse Sertoli cells in the bactericidal testicular defense mechanism mediated by a panel of TLRs. To better characterize the potential role of Sertoli cells in the response against testicular viral infections, we investigated the TLR3 expression and function in these cells. Sertoli cells express TLR3, and under stimulation with the synthetic double-stranded RNA analogue poly (I:C), they produce the proinflammatory molecule ICAM1 and secrete functionally active CCL2 chemokine. Using both pharmacological and genetic approaches, we found that these effects are TLR3-dependent. Moreover, using ELISA, we found that IFNA is constitutively produced and not further inducible, whereas IFNB1 is absent and dramatically induced only by transfected poly (I:C), indicating different control mechanisms underlying IFNA and IFNB1 production. To conclude, poly (I:C) elicits both inflammatory and antiviral responses in Sertoli cells.

TLR Stimulation of Prostate Tumor Cells Induces Chemokine-Mediated Recruitment of Specific Immune Cell Types

TLRs boost antimicrobial response mechanisms by epithelial cells and represent the first line of defense at mucosal sites. In view of these immunomodulatory properties, TLR stimulation may represent a novel means to activate anticancer immune responses. In the present study, the ability of TLR ligands to affect the recruitment of different immune cell populations by human prostate cancer cell lines and the underlying mechanisms were investigated. We showed that LNCaP and DU-145 cells express functionally active TLR3 and TLR5. Treatment with their respective agonists, polyinosinic:polycytidylic acid and flagellin, rapidly triggered NF-κB–dependent upregulation of different inflammatory molecules, as assayed by microarray and ELISA. Furthermore, we demonstrated that conditioned media from polyinosinic:polycytidylic acid- and flagellin-treated LNCaP and DU-145 cells induced the recruitment of different leukocyte subpopulations, suggesting that TLR stimulation is able to activate the earliest step of immune response mediated by soluble factors. Interestingly, the more aggressive cancer cell line PC3 expressed TLR3 and TLR5 but failed to respond to TLR agonists in terms of NF-κB activation and the ability to attract immune effectors. Overall, these data show for the first time that TLR3 and TLR5 stimulation of human prostate cancer cells triggers the production of chemokines, which, in turn, favor the attraction of immune effectors, thereby representing a tool to enhance the efficacy of conventional therapies by stimulating anticancer immune responses.

Mouse Sertoli Cells Display Phenotypical and Functional Traits of Antigen-Presenting Cells in Response to Interferon Gamma

Abstract The testis is regarded as an immunologically privileged site because it tolerates either autoantigenic germ cells or allografts. Because the blood testis barrier represents an incomplete immunological barrier, we have explored whether Sertoli cells, the somatic cells of the seminiferous epithelium, might play an active role in immune evasion. We report data indicating that B7-H1(officially known as CD274)-mediated co-inhibition, an immunomodulatory mechanism based on cell-cell interaction, can be activated in Sertoli cell-lymphocyte cocultures. We have found that, in response to interferon gamma (IFNG), mouse Sertoli cells strongly up-regulate the negative co-stimulatory ligand B7-H1 but remain devoid of positive co-stimulatory molecules. Blockade of B7-H1 on the Sertoli cell surface resulted in enhanced proliferation of CD8+ T cells cocultured with Sertoli cells. Moreover, IFNG-stimulated Sertoli cells were found to express, concurrent with B7-H1, MHC class II. Therefore, we have hypothesized that Sertoli cells could function as nonprofessional tolerogenic antigen-presenting cells by inducing enrichment in regulatory T cells (Tregs) in a mixed T lymphocyte population. Interestingly, we found that coculturing T cells with Sertoli cells can indeed induce an increase in CD4+CD25+(officially known as IL2RA)FOXP3+ Tregs and a decrease in CD4+CD25− T cells, suggesting Sertoli cell-mediated Treg conversion; this process was found to be B7-H1-independent. Altogether these data show that Sertoli cells are potentially capable of down-regulating the local immune response, on one hand by directly inhibiting CD8+ T cell proliferation through B7-H1 and, on the other hand, by inducing an increase in Tregs that might suppress other bystander T cells.

The Fas system in the seminiferous epithelium and its possible extra-testicular role.

The Fas system is involved in the control of immune system homeostasis and nonfunctional Fas system leads to autoimmune disease in mice and humans. The Fas system is a mechanism through which cells expressing Fas ligand (FasL) induce apoptosis of Fas expressing cells. In mouse and rat, the testis represents the main source of constitutive FasL in the body. The roles so far proposed for this molecule in the testis, such as maintenance of immunoprivilege and regulation of physiological germ cell apoptosis, need to be reconsidered as both hypotheses are based on an erroneous cellular location of FasL in the seminiferous epithelium. Recently, we demonstrated that in rodents FasL mRNA is present in germ cells and not in Sertoli cells, and that FasL protein is displayed on the surface of spermatozoa. Here we propose that, for the mouse spermatozoa, the FasL may represent a self‐defence mechanism against lymphocytes present in the female genital tract. To verify this hypothesis, we performed crossings between males gld, with nonfunctional FasL, and syngenic or nonsyngenic females. We observed a significant decrease of litter size in outbred crossings with gld males compared with wild‐type males, suggesting a possible role of FasL in immunoprotection of the sperm in the female genital tract. The possibility that in humans, by analogy with mouse, FasL plays a self‐protective role for the spermatozoon cannot be excluded, and awaits experimental information on the expression of FasL on human sperm cells.

FLIP is expressed in mouse testis and protects germ cells from apoptosis

AbstractApoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIPL) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIPL expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIPL expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIPL expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIPL expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIPL, are sensitive to Fas-mediated apoptosis.These data indicate for the first time that c-FLIPL might control germ cell apoptosis and caspase activity in the adult testis.